Antibiotic a150a

ABSTRACT

Antibiotic A150A, produced by Streptomyces hygroscopicus strain NRRL 3444 under submerged aerobic fermentation conditions in liquid culture medium and recovered from the fermentation broth any mycelium by extraction, has activity against a variety of microorganisms, including bacteria, fungi, and protozoa, and is especially active against some plant pathogens.

United States Patent 11 1 Hamill et al.

Jan. 16, 1973 ANTIBIOTIC A 150A Inventors: Robert L. Hamill, New Ross;Michael E. Haney, Jr., West Lafayette; Marvin M. lloehn, lndianapolis,all of lnd.

Assignee: Eli Lilly and Company, Indianapolis,

Ind.

Filed: May I4, 1971 Appl. No.: l43,344

US. Cl ..424/l22 Int. Cl.. ..A6lk 21/00 Field of Search ..424/l22;195/80 References Cited OTHER PUBLlCATlONS Miller, The Pfizer Handbookof Microbial Metabolities, McGraw-Hill Book Co., lnc., 196], page 580.

' Primary Examiner-Jerome D. Goldberg Altorney-Everett F. Smith andWalter E. Buting [57] ABSTRACT 1 Claim, Drawing Figure ANTIBIOTIC A 150A SUMMARY This invention relates to a novel antibiotic and to a methodfor its production. In particular it relates to a neutral, nitrogenousantibiotic produced by culturing Streptomyces hygroscopicus NRRL 3444under submerged aerobic fermentation conditions in a nutrient culturemedium containing assimilable sources of carbon, nitrogen and inorganicsalts.

The antibiotic of this invention, arbitrarily designated herein asantibiotic Al50A, inhibits the growth of microorganisms which arepathogenic to animal and plant life. Antibiotic Al50A is especiallyuseful in the control of fungal diseases of economically importantplants in particular bean rust and wheat leaf rust.

The antibiotic Al50A is recovered from the fermentation medium byfiltering the mycelium and thereafter extracting the antibiotic activityfrom both the mycelium and the crude filtered broth. The antibiotic isisolated from the extract as a crude oil by evaporating the extract todryness in vacuo. The crude antibiotic Al50A can be purified andobtained crystalline by chromatography over silica gel or aluminafollowed by crystallization from benzene or ether.

DETAILED DESCRIPTION Antibiotic Al50A as isolated is obtained as awhite, crystalline substance having a melting point of about l66-l68C.

Al50A is insoluble in water and hydrocarbons such as hexane. It issoluble in the lower alcohols such as methanol, ethanol, and the like,esters such as methyl acetate, ethyl acetate and amyl acetate, the loweralkyl ketones such as acetone and methyl ethyl ketone, the

lower alkyl ethers such as diethyl ether and di-n-propyl ether, thehalogenated hydrocarbons such as chloroform, and aromatic hydrocarbonssuch as benzene, toluene, and the like.

Antibiotic Al50A is stable from pH 1 to pH 8, but it is unstable abovepH 9.

The specific optical rotation, of Al50A is +66.86 (C=1, methanol).

Electrometric titration of Al50A in 66% aqueous dimethylformamide showsthe absence of titratable groups.

Antibiotic A 1 50A reacts with acetic anhydride in the presence ofpyridine to yield an acetylated derivative. The molecular weight ofAl50A, as calculated from mass spectral data obtained with the acetylderivative, is approximately 735. Microanalysis of Al50A gives thefollowing elemental composition:

65.06 percent carbon 8.87 percent hydrogen 1.89 percent nitrogen 23.39percent oxygen The empirical formula of Al50A as calculated from themicroanalytical and mass spectral data was determined to be C I-I NO Apower X-ray diffraction pattern of crystalline Al50A using vanadiumfiltered chromium radiation and a wavelength value of 2.2896 A forcalculating the interplanar spacings gives the following values:

The infrared absorption spectrum of Al50A in chloroform solution isshown in the accompanying drawing. The following distinguishableabsorption maxima in the spectrum are observable over the range of 2.0to 15.0 microns: 2.92, 3.4, 5.85, 6.3, 7.25, 7.5, 8.15-8.35, 8.6, 8.7,9.32, and 10.29 microns.

Ultraviolet absorption spectra of Al50A show absorption maxima at 274 p.(E, ,,,=85) under basic conditions. No absorption was observed underacidic or neutral conditions.

The paper chromatographic behavior of Al50A is shown by the R; valueslisted in Table 1. The values were obtained in the indicated solventsystems with the use of Whatman No. 1 paper in each instance. Thelocation of the antibiotic on the chromatogram was determined bybioautograph using Sarcina lutea as the detecting organism.

TABLE I Paper Chromatography of Antibiotic Al50A Solvent System R,Va1ueA 1 50A Butanol saturated with water; 0.87

2% p-toluenesulfonic acid Methylisobutylketone saturated with water 0.72

Methylisobutylketcne saturated with water; 0.73

2% p-toluenesulfonic acid Methylisobutylketone saturated with water;0.67

2% piperidine \Vaterzmethanolzacetone 12:3:1)" 0.51

10% aqueous propanol 0.50

Butanolzethanolzwater (13:5:152150) 0.75

R, value is defined as the ratio of the distance traveled by theantibiotic from the origin to the distance traveled by the solvent frontfrom the origin.

"This solution is adjusted to pH 10.5 with NILOH; then the pH is loweredto 7.5 with H,P,O..

Thin layer chromatography of antibiotic Al50A on silica gel plates runin ethyl acetate solvent shows an active factor having an R, value of0.61 as detected by vanillin or sulfuric acid spray. Bioautographs ofthin layer plates may be prepared with the use of sensitive organismssuch as Sarcina luzea.

The novel antibiotic of this invention has an inhibitory action againstthe growthof microorganisms, including bacteria, fungi, and protozoa,which are pathogenic to animal and plant life and is therefore useful insuppressing the growth of such organisms. The minimum inhibitoryconcentrations of Al50A as determined by the agar dilution test arelisted in Table II.

v Ties t Organism TABLE II.

In Vitro Antibiotic Activity of Al50/\ Concentration (meg/ml)Staphylococcus aureus 100.00 Bacillus subtilis 25.00 Mycobacterium avium25.00 Trichaphyton mentagrophytes 6.25 Allernaria solani 6.25 Botrytiscinerea 6.25 Ceratoslomella ulmi 25.00 Cladosporium resinae 100.00 IColletotrichum pisi 6.25 Helminthosporium sativum 100.00 Pullularis sp.100.00 Spicaria divaricata 25.00

Neegleria gruberi (acanthameba) in tissue culture 50.00

When tested in disc plate tests, A150A also produces discernible zonesof inhibition against the microorgan isms Ochromonas malhamensis,Chlorella vulgaris, and Scenedesmus basiliensis.

The antibiotic of this invention, when applied to plants, will inhibitthe development of certain plant diseases. It is active against powderymildew in bean plants, bean rust, anthracnose in cucumbers, rice blast,

and leaf rust in wheat.

The precise manner of application of antibiotic A150A to plants is notcritical. Generally, initial contact of the causative organism is withplant foliage; for this reason, foliar application of antibiotics isoften preferred.

The present method for controlling plant-pathogenic fungi is byapplication of an effective amount of antibiotic A150A to a plantexposed to the fungi. In preparing formulations for such applications,the antibiotic can be modified with water or other liquid carriers,organic solvents, surface-active agents, inert fine ly-divided solids,or inert granular solids. The exact concentration of antibiotic in suchformulations is not critical and will vary with the particular purposefor which the formulation is designed.

In preparing liquid formulations, antibiotic A150A can be compoundedwith a suitable liquid and a surface-active dispersing agent to produceemulsifiable concentrates which can be further diluted with water orother liquid to form spray mixtures. in the preparation of dustcompositions, the antibiotic can be compounded with any of the finelydivided solids or granules typically used in agricultural chemicalformulations.

The antibiotic can be applied to plants in any convenient fashion, suchas by means of hand dusters or sprayers. Application of A150Aformulations to plants can be conveniently carried out with powderclusters, boom Sprayers, high-pressure Sprayers, and spray dusters. Insuch foliar applications, the employed compositions should not containany appreciable amount of phytotoxic diluents. I I

The following examples illustrate the use of antibiotic AlSOA for thecontrol of plant pathogens and will enable those skilled in the art topractice the same. Control of Wheat Leaf Rust Antibiotic AlSOA wasevaluated for the control of wheat leaf rust (Puccinia recondita). Theevaluation was carried out in accordance with the following procedure:4-inch pots of soil were thickly seeded with wheat and held undertypical greenhouse conditions for 6 days.

Minimum Inhibitory '4 The inoculum for the plants was prepared byharvesting uredospores of wheat leaf rust from infected wheat andsuspending the spores in distilled water.

Treating solutions consisted of antibiotic A 1 50A dissolved atconcentrations of 50, 100, 200, and 400 ppm in a mixture of ethanol:l%acetone (1:1) containing 0.1% polyoxyethylene sorbitan monolaurate. Eachsolution was sprayed onto the wheat in two pots and allowed to dry; thewheat was then inoculated with the aqueous spore suspension by spraying.The pots were placed in a moist chamber at 18C for 48 hours, and werethereafter returned to the greenhouse and held under typical greenhouseconditions for 6 days. Controls were sprayed with solvent containing nocompound; otherwise, they were treated identically.

Results of the evaluations were as set forth in the following table,using the disease rating system below:

1 severe 2 moderately severe 3 moderate 4 slight 5 no disease TABLE [11Control of Wheat Leaf Rust Concentration of AISOA in Treating Solution(ppm) Disease Rating 50 4, 3 4, 4+ 200 4+, 4+ 400 5, 4+

Untreated control plants showed extensive symptoms of wheat leaf rust.No phytotoxicity was observed. Control of Bean Rust Antibiotic AlSOA wasalso evaluated for control of bean rust (Uromyces phaseoli var. typica).The evaluation was carried out as follows: Four bean seeds' were plantedin sand in 4-inch pots and held under typical greenhouse conditions for9 days, during which time two of the plants were thinned out.

An inoculum was prepared by suspending 50 mg. of spores of the causativeorganism (harvested from diseased bean plants) in 100 ml. of distilledwater.

The primary leaves of each plant were inoculated with rust spores andthe plants placed in a moist chamber at 18C for 24 hours. The plantswere then removed from the pots, and were placed in test tubescontaining antibiotic Al50A in physiological saline, and held in thegreenhouse for 6 days. Under these conditions, the plants were aeratedfour times daily. Controls consisted of bean plants inoculated withfungus and placed in tubes containing saline without antibiotic.Readings were made in accordance with the disease rating scalepreviously reported above. Averages from several tests are shown in thefollowing table:

TABLE IV Control of Bean Rust Concentration of AlSOA in TreatingSolution (ppm) Disease Rating Untreated control plants showed extensivesymptoms of bean rust. Slight to moderate phytotoxicity was observed inplants treated with the higher levels of A150A.

Antibiotic AOA also exhibits inhibitory activity against powdery mildewin beans, anthracnose in cucumbers, and rice blast when administered asa foliar spray.

The actinomycete used for the production of the antibiotic of thisinvention has been identified as a strain of Streptomyces hygroscopicus(Jensen) Waksman and Henrici. The organism has been deposited with thepermanent culture collection of the Northern Utilization Research andDevelopment Division, Agricultural Research Service, US. Dept. ofAgriculture, Peoria, lll. lts accession number in this collection isNRRL .3444. The strain was isolated from a soil sample collected inKorea by suspending portions of the sample in sterile deionized waterand streaking the suspensions on nutrient agar in Petri plates. Afterincubation at -30 C colonies of the Al50A-producing organism weretransferred to agar slants with a sterile platinum loop. The agar slantswere then incubated to provide a suitable inoculum for the production ofA150A.

The methods employed in the taxonomic studies of the Al50A-producingculture, NRRL 3444, were those recommended for the InternationalStreptomyces Project [Shirling and Gottlieb, Intern. Bull. SystematicBacterioL, 16:313-340 (1966)], together with certain supplementarytests. Results of the taxonomic studies are summarized in the paragraphswhich follow. Color names were assigned according to the Inter-SocietyColor Council National Bureau of Standards (lSCC- NBS) method (Kelly andJudd, the lSCC-NBS Method of Designating Colors and a Dictionary ofColor Names, US. Dept. of Commerce Circ. 553, Washington, DC. 1955).Letters in parentheses refer to color blocks and underlined letters andnumbers to color tabs in the Tresner and Backus color series [Appl.Microbiol. l 11335-338 (1963)]. The Maerz and Paul color blockdesignations [Dictionary McGraw-Hill Color, McGraw Hill Book Co., Inc.New York (1950)] are enclosed in brackets. ISP numbers refer toInternational Streptomyces Project media (available from DifcoLaboratories, Detroit, Mich.). Observations were made followingincubation at C for 14 days unless otherwise noted.

The organism is principally characterized by the formation of tightlycoiled sporophores and by brownish gray aerial mycelium which produce adistinctive hygroscopic character on certain media.

The organism is a member of the Spira section gray series as defined byPridham et al. [AppL MicrobioL, 6:52-79 (1958)] and is also classifiedin the Gray series of the Tresner and Backus system [AppL MicrobioL,11:335-338 (1963)].

brown [13C7]. Aerial mycelium dark gray to black (GY) 3ih; hydroscopic.No soluble pigment.

Growth moderate; reverse olive gray [lSCl Aerial mycelium dark gray (GY)3ih; hygroscopic. No soluble pigment.

Growth abundant; reverse light grayish olive [2lBl Aerial mycelium darkgray to black (GY) 3ih; hygroscopic. No soluble pigment.

Growth abundant; reverse light grayish olive [21B] Aerial lSP Medium No.3

lSP Medium No. 4

IS! Medium No. 5

mycelium yellowish gray to medium gray (GY) g. No soluble pigment.

Tomato Paste-Oatmeal No growth.

Agar

Emerson s Agar Growth fair to moderate; reverse light olive [1414].Aerial mycelium white (W) a. No soluble pigment.

Growth abundant; reverse light olive [22K] Aerial mycelium dark gray toblack (GY) 3H1; hygroscopic. No soluble pigment.

Growth fair to moderate; reverse pale yellow [llC2]. Aerial myceliumwhite (W) a. No soluble pigment.

Growth abundant; reverse light grayish olive (22C! Aerial myceliumbrownish gray (GY) 5th. No soluble pigment.

Growth fair; reverse light yellow brown [1285]. No aerial mycelium. Nosoluble pigment.

Very limited vegetative growth.

Bennett's Agar Czapeks Agar Glucose-Asparagine Agar Tyrosine AgarNutrient Agar Calcium Malate Agar Growth moderate; reverse pale yellowgreen [lOCl No aerial mycelium. No soluble pigment.

Physiology:

Fair growth and sporulation at 26C. Abundant growth and sporulation at30 and 37C.

Temperature Requirements Sparse growth and slight aerial mycelium at49C. No growth at 55C.

Melanin Production Peptone lron Agar Negative.

Tyrptone Yeast Negative.

Extract Broth Skim Milk Response of vegetative color to pH changeNitrate Reduction Coagulation; peptonization Unaffected. Positive.

TABLE V Carbon Utilization of NRRL 3444 Substrate Response L-arabinoseSucrose D-xylose D-fructose Glucose Rhamnose Rafl'inose i-inositolD-mannitol As previously noted, strain NRRL 3444 can be grown in aculture medium to produce antibiotic AA. The culture medium can be anyone of a number of media since, as is evident from the abovedescribedutilization tests, the organism is capable of utilizing energy from avariety of sources. For economy of production, maximum yield, and easeof isolation of the antibiotic, however, certain culture media arepreferable. For example, the media which are useful in the production ofAlSOA include an assimilable source of carbon such as sucrose, fructose,glucose, molasses and the like. The preferred source of carbon issucrose. In addition, employable media contain a source of assimilablenitrogen such as beef extract, peptones (meat and soy), hydrolyzedcasein, yeast extract, amino acid mixtures, and the like. Presentlypreferred sources of nitrogen are peptones (meat and soy).

Mineral salts, for example, those providing calcium, magnesium, sodium,potassium, chloride, sulfate, and carbonate ions, and a source of growthfactors, such as yeast or yeast extract, can be incorporated in themedia with beneficial results.

As is necessary for the growth and development of other microorganisms,essential trace elements should also be included in the culture mediumfor growing the actinomycete employed in this invention. Such traceelements are commonly supplied as impurities incidental to the additionof the other constituents of the medium.

The initial pH of the culture medium can be varied. It has been founddesirable, however, that the initial pH of the medium be between pH 5.5and pH 7.0, and preferably about pH 5.8 to about pH 6.8. As has beenobserved with other actinomycetes, the pH of the medium graduallyincreases throughout the growth period of the organism while A150A isbeing produced, and may attain a level from about pH 7.0 to about pH7.9, the final pH being dependent at least in part on the initial pH ofthe medium, the buffers present in the medium, and the period of timethe organism is permitted to grow.

Submerged, aerobic cultural conditions are the conditions of choice forthe production of A150A; For preparation of relatively small amounts,shake flasks can be employed, but for the preparation of large amountsof the antibiotic, submerged aerobic culture in sterile tanks ispreferred. The medium in the sterile tank can by inoculated with a sporesuspension, but, because of the growth lag experienced when a sporesuspension is used as the inoculum, the vegetative form of the cultureis preferred. By thus avoiding the growth lag, more efficient use of thefermentation equipment is realized. Accordingly, it is desirable firstto produce a vegetative inoculum of the organism by inoculating arelatively small quantity of culture medium with the spore form of theorganism. When a young, active vegetative inoculum has been obtained, itis transferred aseptically to the large tank. The medium in which thevegetative inoculum is produced can be either the same as or differentfrom the medium utilized for the large scale production of A150A.

The organisms grow best at temperatures in the range of 30 to 37C.Optimal Al50A production appears to occur at a temperature of about 30C.

As is customary in aerobic, submerged culture processes, sterile airisblown through the culture medium. For efficient growth of the organismand AlSOA production, the volume of air employed in the tank productionof Al50A preferably is 0.5 volumes of air per minute per volume ofculture medium. Efficient growth and optimal yields of AA are obtainedwhen the volume of air is at least 0.3 volumes of air per minute pervolume of culture medium.

The concentration of Al5OA activity in the culture medium can readily befollowed during the fermentation period by testing samples of theculture broth for their inhibitory activity against the growthofmicroorganisms known to be inhibited in the presence of antibioticAlSOA. The use of Sarcina later: and Bacillus subtilis. have been foundto be suitable for this purpose. The testing can be carried out by .theknown turbidimetric or disc-plate methods.

In general, maximum production of the antibiotic occurs within aboutthree to four days after inoculation of the culture medium whensubmerged aerobic culture conditions are employed.

Antibiotic AlSOA can berecovered from the fermentation mixture accordingto methods commonly employed for the isolation of antibiotics. Recoverymethods employing extractive and chromatographic techniques for examplecan be used in the recovery and isolation of antibiotic A150A.

Accordingly the mycelium and undissolved solids are removed from thefermentation broth by conventional means such as filtration orcentrifugation.

The antibiotic activity associated with the mycelium is extractedtherefrom with a suitable solvent such as methanol, ethanol or acetone.Although in general small amounts of the antibiotic are recovered fromthe mycelium the amount obtained thereby varies. Such amounts justifyrecovery in the interest of economy.

The major amount of the antibiotic activity is found in the crudefiltered broth and can be recovered by extracting the broth with asuitable solvent. Acceptable extraction solvents include the commonesters'such as ethyl acetate or amyl acetate, and the chlorinatedhydrocarbon solvents such as chloroform and methylene dichloride. Thepreferred solvent is chloroform.

Conveniently, the mycelium extract is added to the crude filtered broth.The pH of the ,broth is then adjusted to about pI-l3 by the addition ofa mineral acid and preferably with 6N hydrochloric acid. The acidifiedbroth is then extracted with chloroform to recover the total antibiotic.activity produced in the fermentation.

The chloroform extract is then decolorized and partially purified bypassing the extract over a column packed with activated carbon. Theantibiotic extract is then evaporated to dryness to yield antibioticAISOA as a crude oil.

The antibiotic can be further purified and obtained crystalline bychromatography over a suitable adsorbent. Chromatographic adsorbentssuch as silica and alumina can beemployed in the purification of AlSOA.

Accordingly, the crude antibiotic A150A oil is dissolved in a suitablesolvent such as ethyl acetate and the solution added to a column packedwith silica gel. The antibiotic can be eluted with ethyl acetate orother solvents of like polarity..The eluate fractions containingantibiotic activity are combined and evaporated to dryness to obtain thepurified A150Aantibiotic as a solid amorphous residue.

The solid amorphous antibiotic can be obtained crystalline bycrystallization from a suitable solvent, for example, ether or benzene.7

Alternatively, the crude antibiotic Al50A obtained as an oil from thebroth extract can be obtained in an impure solid form by dissolving theoil in a solvent such as ether or benzene and diluting the solution witha hydrocarbon solvent such as hexane to precipitate the antibiotic as anon-crystalline solid. The antibiotic solid thus obtained can be furtherpurified by chromatography over a suitable adsorbent such as silica gelas described previously.

The crude solid antibiotic is suitable for certain uses, for example,for incorporation in agricultural formulations for the treatment ofdiseased plants.

This invention is further illustrated by the following examples, but isnot limited thereby.

Example 1 A. Shake-flask fermentation of Al50A The A l SOA-producingculture is prepared and maintained on an agar slant having the followingcomposition:

Glucose 10.0 g Yeast extract 10.0 g Beef extract 4.0 g Peptone 4.0 gNaCl 2.5 g Agar 20.0 g Deionized water 1 liter Glucose Peptone Beefextract NaCl Hydrolyzed casein Deionized water Enzymatic hydrolyzedcasein (N-Z Amine, Sheffield Chemical Division of Natural DairyProducts, Norwich, N.Y.)

The inoculated vegetative medium is incubated for 48 hours at 30C on areciprocal shaker having a 2-inch stroke at 108 strokes per minute. Al-ml portion of the resulting culture is then employed to inoculate 200ml of sterilized production medium contained in a liter Erlenmeyer flaskand having the following composition:

Glucose l Edible molasses 2 Peptone (Difco Bacto) 5. CaCO, 2 Tap water lThe inoculated medium is allowed to ferment for 48 hours at 30C on areciprocal shaker operating at 108 strokes per minute. The terminal pHis 7.0.

B. Tank fermentation of A 1 50A The A l SOA-producing culture isprepared and maintained on an agar slant having the followingcomposition:

Sucrose 10.0 g Casamino acids (Difco) 2.0 g Malt extract 0.25 g Yeastextract 0. 2 5 g KC! 0 5 E 5 Mes -m g FeSO -7H,O 0.01 g KH,PO4 1.0 gMeer Agar (Washed 3X) 20.0 g Deionized water I liter l0 Meer Co., 318 W.46the St., New York, N.Y.

The pH of the medium was not adjusted; th sterilization, the pHis 5.8.

The slant is inoculated with the AISOA-producing culture, NRRL 3444, andincubated at 34C for 7-10 days. The sporulated slant is covered with asmall amount of sterile deionized water and gently scraped to provide anaqueous spore suspension.

Each slant is used to inoculate six flasks (50 ml/250 ml flask) ofsterile vegetative culture medium having the following composition:

Glucose 10.0 g Sucrose 30.0 g Peptone 5.0 g Hydrolyzed casein 5.0 g

Hydrolyzed yeast 5.0 g Beef extract 5.0 g CaCO 2.0 g Tap water 1 literHydrolyzed yeast Amber BYF 300, Amber Laboratories, Juneau,

Wisconsin.

The pH of the medium is unadjusted; after sterilization, the pH is 6.5.

The inoculated medium is allowed to ferment for 48 hours at 30C on arotary shaker operating at 250 R.P.M. A lO-ml portion of the resultingculture is used to inoculate 200 ml of sterilized second-stage growthmedium contained in a liter flask and having the same composition asthat described above.

The inoculated medium is allowed to ferment for 48 hours at 30C on areciprocal shaker operating at 250 R.P.M. A 200 ml portion of theresulting culture is used to inoculate 25 liters of the following mediumin a 40- liter fermentor:

Percent Dow-Corning Antifoam A" 0.02 Glucose 1.5 Sucrose 3.0 Peptone l.0Soybean grits 1.0

50 Molasses 2.0 CaCO 0.2 Tap water 25 liters filtered with 3% Hyflosupercel filter aid. The mycelial cake is extracted by stirring with 8liters of methanol for one-half hour.

The resulting methanolic extract is concentrated in vacuo to remove themethanol. The filtrate and extract are combined, and the pH was adjustedto pH 3 with 6N hydrochloric acid. The. acidified crude broth wasextracted by stirring with 20 liters of chloroform for 15 minutes. Thechloroform extract is concentrated to a small volume and passed througha column packed with carbon to remove color. The effluent isconcentrated in vacuo to an oil. After the oil is dissolved in diethylether, ten volumes of petroleum ether are added to precipitate theactive antibiotic factor. The precipitate is filtered, washed with coldhexane, and dried in vacuo. The precipitate is dissolved in 14 ml. ofethyl acetate and the solution is passed over a column packed with amixture of 40 g. of silica gel H (E. Merck A. G., Darmstadt, Germany)and g. of Hyflo supercel in ethyl acetate. The column is then developedwith ethyl acetate. The eluates are monitored by thin-layerchromatography and microbial assay. The active fractions are combinedand the total evaporated to dryness in vacuo to obtain antibiotic A150Aas an amorphous solid. The residue is crystallized from warm benzene orfrom ether to obtain antibiotic A150A as a white crystalline compoundmelting at about 166-1 68C.

We claim:

1. The antibiotic A150A, said antibiotic being characterized in itscrystalline form as a white crystalline compound melting at about 166 to168 C. which is substantially insoluble in water and hexane and is soluble in methanol, ethanol, ethyl acetate, acetone, diethyl ether, benzeneand chloroform; which has an apparent molecular weight of 735 asdetermined by mass spectral data; which has an approximate elementalcomposition of 65.06 percent carbon, 8.87 percent hydrogen, 1.89 percentnitrogen and 23.39 percent oxygen as determined by .r'nicroanalysis;which has a specific rotation [01],, 66.86 (C 1, methanol) which isnon-titratable in 66 percent aqueous dimethylformamide; which has thefollowing absorption maxima in its infrared absorption spectrum over therange of 2.0 to 15.0 microns: 2.92, 3.4, 5.85, 6.3, 7.25, 7.5,8.15-8.35, 8.6, 8.7, 9.32, and 10.29 microns; and which shows anabsorption maximum in its ultraviolet spectrum at 274 millimicrons of Eat basic pH.

